Over 180,000 Americans are living with solid organ transplants. Each year over 2000 heart transplants, including over 300 pediatric heart transplants are performed resulting in over 20,000 living heart transplant recipients in the U.S. alone. One year survival following heart transplantation has improved to over 90%; however, 10 year survival remains less than 60%. With improvement in operative and postoperative management, rejection has become the major determinant of outcome. Earlier detection of rejection facilitates earlier treatment, which improves both early and late function and survival. The gold standard in surveillance of cardiac allograft rejection remains catheter based endomyocardial biopsy (EMB). To detect rejection early, surveillance biopsies must be performed at regular intervals; however, EMB is both expensive (over $4000) and invasive (major complications 1.9%). It is estimated that over 30,000 surveillance biopsies are performed each year identifying 3,000 rejections with over 300 major complications. There is a tremendous need for a highly sensitive, non-invasive method that reliably detects patients with rejection. This proposal will have high impact on the problem of rejection surveillance in cardiac transplantation promoting earlier detection, earlier treatment, and improved long term outcomes. Donor specific cf-DNA increases in patients with solid organ rejection. We have completed a blinded pilot study in 38 pediatric and adult heart transplant patients utilizing a novel, highly accurate, targeted sequencing approach which precisely quantifies the amount of donor specific cell free DNA (cf-DNA) and total cf-DNA in recipient plasma. This data established a threshold below which no sample had rejection (0/51), and above which all patients with ISHLT grade e2 rejection (6/6) were detected (100% sensitivity). We have assembled a consortium of 6 major pediatric and adult transplant centers for a blinded multi-center study with independent pathology and echo core labs to test this extremely promising, cost effective, and innovative approach. We will first determine a threshold and develop a predictive model and then validate both on a prospective and blinded sample set of 240 new pediatric and adult transplant recipients followed longitudinally for at least one full year. Finally we will explore the relationships between major pathologic and clinical observations and levels of donor specific and total cf-DNA. This method holds promise to enable the accurate and cost effective non- invasive determination of rejection in all solid organ transplantation.